Composition and Methods for the Treatment of Degenerative Retinal Conditions

ABSTRACT

The present invention is directed to compositions and methods for the treatment of degenerative retinal conditions. According to a general aspect, the present invention is directed to inflammatory mediators, preferably components or substrates of the NLRP 3 -inflammasome, for use in the treatment of degenerative retinal conditions involving drusen and anaphylatoxin-induced choroidal-neovascularisation. The invention is also directed to a method for the treatment of degenerative retinal conditions involving drusen and anaphylatoxin-induced choroidal-neovascularisation and to recombinant vectors and recombinant proteins for use in such methods. The present invention also provides a method for determining the risk of developing or monitoring the progression of diseases involving drusen and anaphylatoxin-induced choroidal neo-vascularisation.

The present invention is directed to compositions and methods for thetreatment of degenerative retinal conditions.

Specifically, the present invention is directed to compositions andmethods for treating degenerative retinal conditions involving thedrusen and anaphylatoxin-induced choroidal neo-vascularisation and tothe use of gene delivery vectors which direct the expression of selectedgene products, including inflammatory mediators, components orsubstrates of the NLRP3-inflammasome, suitable for treating orpreventing degenerative retinal conditions involving the drusen andanaphylatoxin-induced choroidal neo-vascularisation, includingpathologically-induced CNV. Preferably, the degenerative retinalconditions involving the drusen and anaphylatoxin-induced choroidalneo-vascularisation is age-related macular degeneration.

In the developed world, age-related macular degeneration (AMD) is themost prevalent cause of legal blindness in older individuals. AMD is aprogressive disease, characterized by the accumulation of focalextracellular deposits on Bruch's membrane below the retinal pigmentepithelium (RPE) in the macula, which is recognized in an eyeexamination as drusen. Drusen accumulation is the major pathologicalhallmark common to both dry and wet AMD. The presence of drusen in themacula, the density of the deposits and the area covered by thismaterial represent early stages in the AMD disease process. Individualswith drusen are considered at risk for progressing to the end-stageblinding forms of AMD. Geographic atrophy (GA), the end stage of theatrophic “dry” form of AMD, culminates in vision loss following focaldegeneration of the RPE below the fovea. Without the RPE, the fovealcone photoreceptors degenerate, causing central retinal blindness.Choroidal neovascularization (CNV) characterizes the end stage of theexudative “wet” form of AMD, with new blood vessels breaking throughBruch's membrane/RPE that hemorrhage, causing a blood clot to formbetween the RPE and foveal photoreceptors resulting in immediateblindness.

The disease, AMD, is classically multi-factorial, with bothenvironmental and genetic factors involved. Sequence variants associatedwith disease susceptibility have now been characterized in a growingnumber of immune regulated genes. Activation of complement on ocularsurfaces is thought to play a major role in the early disease process,resulting in drusen deposition. However, the mechanisms involved in theinitiation of the inflammatory responses observed in the eyes of AMDsubjects are still unresolved.

Drusen deposits are particulate protein aggregates, extra-cellular innature, characteristics of known activators of the sterile inflammatoryresponse mediated by NACHT, LRR and PYD domains-containing protein 3(NLRP3). NLRP3 acts as a receptor for “danger” signals such as ATP, uricacid crystals, amyloid-like structures and mitochondrial dysfunction,which activate the inflammasome comprising NLRP3, Apoptosis-associatedSpeck-like domain containing a Caspase-recruitment domain (ASC) andpro-caspase-1 and resulting in the cleavage of pro-IL-1β and pro-IL-18,into their mature pro-inflammatory forms. Furthermore, excessive drusenaccumulation can disrupt the adjacent RPE cells which subsequently dieby necrosis a cellular process now known to activate the NLRP3inflammasome.

Current antibody-based therapies target advanced forms of AMD byinhibiting the bioactivity of VEGF. However, direct and regularintraocular injection of these monoclonal antibodies (Lucentis® andAvastin®) carry the risk of retinal detachment, haemorrhage andinfection. No FDA-approved treatments are available for dry maculardegeneration, although a few now are in clinical trials. Thus, thegeneration of new AMD therapies is important commercially.

From a clinical perspective, while inflammatory processes have long beenassociated with AMD pathology and disease development, we suggest thatglobal inhibition of inflammation in the retina in the case of wet AMDwould not be a sound therapy. Lending strength to our observations, theresults of recent clinical trials of Infliximab (Remicade®) inindividuals with wet AMD showed that in more than 50% of these subjects,symptoms were greatly exacerbated.

IL-18 is a molecule with a wide-ranging variety of functions, many ofwhich are dichotomous opposites of each other depending on theenvironment in which IL-18 is released. This is the case with regards tothe role of IL-18 in regulating angiogenesis or blood vessel growth. Forexample, IL-18 has been shown to have a pro-angiogenic effect in a modelof rheumatoid arthritis (Park et al, 2001, Evidence of IL-18 as a novelangiogenic mediator. Journal of Immunology 167: 1644-1653) whereas ithas been shown to be anti-angiogenic in herpetic stromal keratitis (KimB et al, 2006, Application of plasmid DNA encoding IL-18 diminishesdevelopment of herpetic stromal keratitis by antiangiogenic effects.Journal of Immunology 175:509-516). The reason for the discrepancybetween the role of IL-18 as an angiogenic agent versus its role as anangiostatic agent remains unclear but is in part determined by thespecific vascular beds involved.

Qiao et al (Interleukin-18 Regulates Pathological IntraocularNeovascularaization. Journal of Leukocyte Biology. Volume 81, April2007, 1012-1021) uses an oxygen-induced model of retinopathy ofprematurity, which triggers the development of intraocularneovascularization. Intraocular neovascularization involvesneovascularization of the blood vessel lining the inner retina. Qiao etal administered recombinant IL-18 to C57BL/6 mice during the developmentof oxygen induced retinopathy, and found no inhibition ofneovascularization. Qiao et al concluded that IL-18 regulatesintraocular retinal neovascularization by promoting its regressionrather than inhibiting its development.

The present invention is directed to a new and improved therapy fordegenerative retinal conditions involving the drusen andanaphylatoxin-induced choroidal neo-vascularisation and/orpathologically-induced CNV, preferably choroidal neo-vascularisationassociated with AMD, more preferably wet AMD.

The choroid is a vascular bed which is extraoccular and completelyseparate to the retina. It does not act in the same way as the retinalintraocular vasculature. Choroidal neovascularization (CNV) is thecreation of new blood vessels in the choroid layer of the eye. This is acommon symptom of the degenerative maculopathy wet AMD (age-relatedmacular degeneration) and is distinct from retinal neovascularization(intraocular) of Qiao et al.

In this specification, the term choroidal neo-vascularisation (CNV)ideally does not encompass retinal neovascularization (intraocular).Intraocular neovascularization involves neovascularization of the bloodvessel lining the inner retina. This is distinct to choroidalneovascularization which involves the choroids, a vascular bed which isextraoccular and completely separate to the retina. Ideally, in thisspecifciation the use of IL-18 for intraocular neovascularization isexcluded.

In this specification, the term “NLRP3 inflammasome, component orsubstrate” will be understood to cover NALP3, ASC and pro-caspase-1 andpro-IL-18 and IL-18. When NLRP3, ASC and pro-caspase-1 oligomerise theyproduce active caspase-1 which then cleaves pro-IL18 to mature Il-18.Thus, pro-IL18 is a component of the inflammasome by virtue of the factthat it is a substrate of caspase-1. The 24 KDa inactive precursor ofIL-18 is referred to as “pro-IL-18” and 18KDa mature IL-18 is referredto as “IL-18” “or pro-inflammatory IL-18”. NLRP3 and NALP3 are usedinterchangeably in this specification and refer to the same inflammasomecomponent. In a preferred embodiment, the NLRP3 inflammasome, componentor substrate is mature Il-18, referred to as “Il-18”. It will beunderstood that reference to IL-18 also means the nucleotide sequencedefined by NCBI Accession no. NM_(—)001562.3 and the protein sequencedefined by NCBI Accession no. NP_(—)001553.1. It will also be understoodthat reference to delivery of IL-18 also covers the delivery ofrecombinant IL-18 (rIL-18), for example locally via intra-ocularinjection or systemically via intravenous injection.

We have shown that drusen isolated from donor AMD eyes activates theNLRP3-inflammasome, causing secretion of IL-18 and IL-18. Drusencomponent C1Q, also activates the NLRP3-inflammasome. Moreover,oxidative-stress related protein-modification carboxyethyl-pyrrole(CEP), a biomarker of AMD primes the inflammasome. We have found thatcleaved caspase-1 and NLRP3 in activated macrophages in the retina ofCEP-MSA-immunised mice, which models a dry AMD-like pathology. We showthat laser induced choroidal-neovascularisation (CNV), an acceptedanimal model of wet AMD, is exacerbated in Nlrp3^(-/-), but notIl1r1^(-/-) mice, directly implicating IL-18 in regulating CNVdevelopment. These findings are indicative of a protective role forNLRP3 and IL-18 in the progression of AMD.

It is our opinion that NLRP3 and it's components are directly implicatedas a protective agent against the major disease pathology of AMD. Thus,strategies aimed at producing or delivering IL-18 to the eye, may provebeneficial in preventing the progression of CNV, especially in thecontext of wet AMD.

According to a first general aspect of the invention, there are providedinflammatory mediators, preferably components or substrates of theNLRP3-inflammasome, for use in the treatment of degenerative retinalconditions involving the drusen and anaphylatoxin-inducedchoroidal-neovascularisation.

Retinal (intraocular) neovascularization and choroidal (extraocular)neovascularization of the present invention are distinct conditions ofthe vasculature. We have found that IL-18 suppresses the promotion ofchoroidal neovascularisation.

Ideally, the components or substrates of the NLRP3-inflammasome isIL-18, including recombinant IL-18.

Ideally, the retinal condition inludes pathologically-induced CNV, suchas choroidal-neovascularisation (CNV) associated with AMD, preferablydry and/or wet AMD, more preferably wet AMD.

According to this general aspect of the invention, the inflammatorymediators, components or substrates may be used in controlling,maintaining or stimulating Interleukin-18 (IL-18) expression in asubject at risk of developing age-related macular degeneration andprevent the progression of CNV, especially in the context of wet AMD.

It will be understood that up-stream components of the inflammasome mayalso be used. These components include NLRP3, ASC or pro-caspase-1,pro-IL-18 etc. However, delivery of IL-18 or recombinant IL-18 ispreferred.

Ideally, the inflammatory mediators, components or substrates, includingInterleukin-18 (IL-18) are administered prior to the development ofneo-vascular disease or in early-stage neo-vascular disease.

Activation of the NLRP3 inflammasome by drusen suggest that a balancemay exist, whereby a certain focal level of drusen is tolerated due toits ability to induce IL-18 which in turn may act as an anti-angiogeniceffector, maintaining choroidal homeostasis in an inflammatorymicro-environment. It is likely that once a critical level of drusenaccumulates, its protective role is negated by excessive damage to thesurrounding tissues. Importantly, we have demonstrated thatdrusen-inducible inflammatory mediators are protective against CNVdevelopment and that it is the resultant NLRP3 mediated elevation ofIL-18 that prevents the downstream production of VEGF. Moreover, IL-18has been shown not to play a role in the development of experimentaluveitis, a more conventional model of inflammation.

According to one embodiment the degenerative retinal condition isage-related macular degeneration. It will be understood that theage-related macular degeneration referred through throughout may be wetage-related macular degeneration or dry age-related maculardegeneration.

According to a preferred embodiment of the invention, there is providedInterleukin-18 (Il-18) or recombinant IL-18 for use in controllingchoroidal-neovascularisation (CNV) in a patient at risk of developingwet age-related macular degeneration (AMD). The resultant NLRP3 mediatedelevation of IL-18 prevents the downstream production of VEGF which wehave found is protective against CNV development.

All references to IL-18 herein should be understood to also refer torecombinant IL-18 (rIL-18).

According to a general aspect of the invention, IL-18 or recombinantIL-18 may be administered to result in and provide a local effect or asystemic effect.

IL-18 or recombinant IL-18 protein may be administered per se. IL-18 orrecombinant IL-18 may also be administered in the form of apharmaceutical composition. Optionally, IL-18 may be administered with apharmaceutically acceptable adjuvant. Such adjuvants may includecarriers, diluents, and excipents such as sterile water and oil. Furtheradjuvants, excipients and auxiliary substances are listed below.

It will be understood that administration may be carried out via avariety of routes. These routes are designed to provide a local orsystemic effect as required. These routes include, but are not limitedto, oral, topical, pulmonary, rectal, subcutaneous, intradermal,intranasal, intracranial, intramuscular, intraocular, or intra-articularinjection, and the like. The most typical route of administration isintravenous followed by subcutaneous, although other routes can beequally effective. Intramuscular injection can also be performed in thearm or leg muscles.

In some methods, IL-18 may be injected directly into a particulartissue. In other embodiments, administration may be as part of asustained-release composition.

For parenteral administration, IL-18 may be administered as injectabledosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier thatcan be a sterile liquid such as water, oils, saline, glycerol, orethanol. Additionally, auxiliary substances, such as -wetting oremulsifying agents, surfactants, pH buffering substances and the likecan be present in compositions. Other components of pharmaceuticalcompositions are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, and mineral oil. Ingeneral, glycols such as propylene glycol or polyethylene glycol aresuitable liquid carriers, particularly for injectable solutions.

IL-18 can also be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained-release of the protein IL-18.

Typically, compositions may be prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced delivery.

Oral formulations may take the form of solutions, suspensions, tablets,pills, capsules, sustained-release formulations or powders. Topicalapplication can result in transdermal or intradermal delivery.Alternatively, transdermal delivery can be achieved using a skin patch.

Ideally, the above administration modes result in delivery of IL-18 tothe eye, preferably to the retina, more preferably to the choroid layer.

According to a preferred embodiment of the invention, local deliverymeans include direct injection of IL-18, rIL-18 or pharmaceutialcomposition comprising IL-18/rIL-18, to the eye, such as intra-ocularinjection. Injections may be sub-retinal, made into the vitreous of theeye (intra-vitreal), behind the eye (retrobulbar), below the conjunctiva(subconjunctival) or under tenon's capsule (subtenons).

According to another preferred embodiment, pro-inflammatoryInterleukin-18 (IL-18), rIL-18 or pharmaceutial composition comprisingIL-18/rIL-18, may be delivered to the subject systemically. Systemicdelivery means may include parenteral or enteral means and essentiallyencompass all non-local delivery means. Ideally, systemic delivery meansinclude injection, direct injection, and/or viral mediated delivery.

According to a preferred embodiment, systemic injection techniquesinclude intravenous delivery by intravenous injection. Intravenousinjection may be to a peripheral vein of the subject. For thisadministration route, IL-18, rIL-18 or a pharmaceutial compositioncomprising IL-18/rIL-18 is adminstered/injected directly into the bloodstream of the subject.

Preferably, IL-18 is delivered to the retina systemically, by directinjection, such as intravenous injection and/or by viral mediateddelivery.

Viral mediated delivery techniques include adeno-associated virus (AAV),adenovirus or lentivirus gene delivery vector. Optionally, viralmediated delivery techniques, including AAV therapy, may be appliedup-stream of inflammasome components by introducing NLRP3, ASC orpro-caspase-1.

According to another preferred embodiment of the invention, there isprovided a recombinant viral gene delivery vector which directs theexpression of pro-IL-18 or IL-18 in a form suitable for the treatment ofchoroidal, neo-vascularisation.

The recombinant viral gene delivery vector may be in a form foradministration systemically, for example by intravenous administration.

The recombinant viral gene delivery vector may also be in a form foradministration to the eye of a subject at risk of developing AMD,preferably wet AMD.

For example, the vector may be in a form suitable for delivery to theeye via intraocular injection, including sub-retinal or intra-vitrealinjection.

The viral vector may be an adeno-associated virus (AAV), adenovirus orlentivirus gene delivery vector. Ideally, delivery of the gene deliveryvector is via sub-retinal injection where, for example, a small amountof fluid is injected underneath the retina. This has an advantage interm of long term disease management following a single administration.The vector may also be optimized for intra-vitreal administration insubjects.

According to a most preferred embodiment of the invention, there isprovided a new gene therapy for degenerative retinal conditionsinvolving the drusen and anaphylatoxin-inducedchoroidal-neovascularisation, preferably wet AMD, involving the use ofviral, preferably AAV, mediated delivery of IL-18 to a subject tocontrol, maintain or stimulate Interleukin-18 (IL-18) expression in asubject at risk of developing a degenerative retinal conditionsinvolving the drusen and anaphylatoxin-inducedchoroidal-neovascularisation, such as age-related macular degeneration(AMD).

According to this preferred embodiment, there is provided a recombinantadeno-associated virus (AAV) gene delivery vector which directs theexpression of IL-18 suitable for administration to the eye of a subjectat risk of developing AMD, preferably wet AMD.

The AAV vector comprises nucleotides encoding Il-18 gene. The AAV may beany one of AAV serotype 1 to 11, preferably serotypes 2, 8 or 9.Ideally, the IL-18 gene is flanked by AAV inverted terminal repeats(ITR).

Many different promoters may be used. These are ideally cell specificpromoters. These may include, retinal pigment epithelial cell (RPE)specific promoters such as CRALBP or RPE65, endothelial cell specificpromoters such as Tie-2 or claudin-5, or photoreceptor specific promoterincluding rhodopsin.

AAV is the main viral vector used for eye diseases as it is veryefficient at transducing retinal cells. It is also non-immunogenic whichis very important from a clinical point of view. AAV technology hasemerged as a very safe means of delivering genes to host tissue,especially tissues of the central nervous system such as the retina. Incontrast to adenoviruses which can integrate into the host genome, AAV'swill infect cells and subsequently their genetic material will reside inthe nucleus as episomal DNA. This factor is likely key to theirexcellent safety profile and low immunogenicity. In tandem, AAVtechnology is now being used in a range of clinical trials forParkinson's disease, Alzheimer's disease and muscular dystrophy and inrelation to this proposal, AAV's are now well established for retinaluse, with numerous clinical trials on-going for the treatment of therecessive form of Retinitis pigmentosa termed Leber's CongenitalAmaurosis (LCA).

To date, 11 AAV serotypes have been described in the literature and thekey differences in these serotypes relates to the target cells that theycan infect. AAV-2, AAV-8 and AAV-9 have been found to transduce RPEcells effectively. Thus, the present invention may preferably involvethe use of AAV-2, AAV-8 and/or AAV-9.

According to one preferred embodiment, the invention involvesconstructing a vector (e.g. AAV vector such as AAV serotypes −2, −8 and−9) by cloning a gene encoding 1L18 including 5′ and 3′ UTRs andintroducing this gene into a vector such as to incorporate left andright AAV inverted terminal repeats (L-ITR and R-ITR).

According to a second aspect of the invention, there is provided amethod of treating degenerative retinal conditions involving drusen andanaphylatoxin-induced choroidal-neovascularisation comprisingadministering inflammatory mediators, components or substrates of theNLRP3-inflammasome to a subject in need of treatment.

According to a preferred embodiment, there is provided a method fortreating wet age-related macular degeneration (AMD) comprisingadministering Interleukin-18 (Il-18) to a subject at risk of developingage-related macular degeneration. IL-18 may be administered to provide alocal or systemic effect.

According to a preferred embodiment, local delivery means include directintra-ocular injection. Injections may be sub-retinal, made into thevitreous of the eye (intra-vitreal), behind the eye (retrobulbar), belowthe conjunctiva (subconjunctival) or under tenon's capsule (subtenons).

According to another preferred embodiment, pro-inflammatoryInterleukin-18 (IL-18) may be delivered to the subject systemically.Systemic delivery means may include parenteral or enteral means andessentially encompass all non-local delivery means. Ideally, systemicdelivery means include injection, direct injection, and/or viralmediated delivery. According to a most preferred embodiment, systemicinjection techniques include intravenous delivery by intravenousinjection. Intravenous injection may be to a peripheral vein of thesubject. For this administration route, recombinant IL-18 isadminstered/injected directly into the blood stream of the subject.

According to a third aspect of the invention, there is provided the useof NLRP3-Inflammasome induced mediators, preferably pro-inflammatoryIL-18 (Il-18), as a biomarker to indicate the risk of developingdiseases involving drusen and anaphylatoxin-induced choroidalneo-vascularisation, such as wet or dry age-related maculardegeneration.

According to a fourth aspect of the invention, there is provided amethod for determining the risk of developing or monitoring theprogression of diseases involving drusen and anaphylatoxin-inducedchoroidal neo-vascularisation, such as wet or dry age-related maculardegeneration, in a subject, using inflammasome induced mediators,preferably pro-inflammatory IL-18 (IL-18), as a biomarker the methodcomprising:

-   -   obtaining the level of circulating IL-18 and/or IL-18 binding        protein levels in a subject;    -   comparing the level of IL-18 and/or IL-18 binding protein levels        to a reference, wherein the subject's risk of development or        progression of the disease drusen and anaphylatoxin-induced        choroidal-neovascularisation is based upon the level of IL-18        levels and/or IL-18 binding protein in comparison to a        reference.

It will be understood that the ratio of IL-18 and its binding protein(IL-18 binding protein) is conventionally used as a measure of variationfrom the normal reference, although IL-18 levels may be used alone.

The invention will now be described in relation to the followingnon-limiting figures and examples.

FIG. 1: Drusen activates the NLRP3 inflammasome: (a) Fundus photographyfrom non-smoking un-affected, dry and wet AMD-affected individuals. (b)Drusen fragments in a range of sizes from just under 500 μm tosub-microscopic sized particles. (c) SDS-PAGE analysis of a Bruch'smembrane (BM)/drusen preparation. (d) Live cell imaging of immortalisedBL6 BMDMs stably expressing yellow-fluorescent protein labelled-ASC(YFP-ASC). Cells were primed for 3 hrs with LPS followed by treatmentwith 250 ng ml⁻¹ drusen for a further 2 hrs, Poly(dAdT) was used as apositive control. Oligomerisation of ASC-YFP was observed by speckformation, original magnification ×60. (e, f) Production of IL-1β andIL-18 was measured by ELISA in Human PBMC primed overnight with 100 ngml⁻¹ LPS and subsequently treated for 7 hours with increasing doses ofthe drusen preparation (250 ng ml⁻¹ and 500ng ml⁻¹) (***P≦0.0001). (g)Western Blot of cleavage products of caspase-1 following treatment ofTHP-1 cells with drusen. (h) Production of IL-1β (left hand panel) andIL-6 (right hand panel) as measured by ELISA in wild-type (WT) (bluebars) and NLRP3-deficient (Nlrp3^(-/-)) (red bars) bone marrow-derivedmacrophages (BMDM's) after treatment with increasing doses of drusen(***P≦0.0001) (i) Production of IL-1β (left hand panel) and TNF (righthand panel) as measured by ELISA in WT (blue bars) and Nlrp3^(-/-)) (redbars) bone marrow-derived dendritic cells (BMDC's) after treatment withincreasing doses of drusen (*** P≦0.0001). All ELISA data arerepresentative of a minimum of 3 separate experiments carried out intriplicate.

FIG. 2: CEP, a component of Drusen can prime the NLRP3 inflammasome: (a)Production of IL-1β in Human PBMC primed with LPS, CEP-adducted albumin(CEP-HSA) or HSA in increasing doses (50 and 100 μg ml⁻¹) andsubsequently treated with 5 mM ATP. (b) IL-1β production in WT andNlrp3^(-/-) BMDMs primed with CEP-HSA and then activated with either ATPor Poly(dAdT). (c, d) IL-1β and IL-6 production in WT or Tlr2^(-/-)BMDMs primed with HSA or CEP-HSA, activated with ATP or left un-treated.(e,f) IL-1β and TNFα production were measured in C3H/HeN BMDMs (WT) orC3H/HeJ BMDMs (which contain a mutant TLR4) primed with either LPS orCEP and activated with ATP. (g) Live cell imaging of immortalised BL6BMDMs stably expressing YFP-ASC. Cells were primed for 3 h with CEP-HSA(top panel) or HSA (bottom panel) followed by treatment with 250 ng/mlDrusen for a further 2 hrs. Oligomerisation of ASC-YFP was observed byspeck formation, original magnification ×60. All ELISA data arerepresentative of a minimum of 3 separate experiments carried out intriplicate.

FIG. 3: Complement factor C1Q, a component of Drusen, activates theNLRP3 inflammasome: (a) Production of IL-1β (left panel) and TNFα (rightpanel) in BMDMs primed with CEP for 3 h and activated for 6 h with C1qat increasing doses. (b) Western blot of caspase-1 cleavage products inTHP1 cells primed with LPS and treated with increasing doses of C1Q. (c)Live cell imaging of immortalised BL6 BMDMs stably expressing YFP-ASC.Cells were primed for 3 hrs with either LPS (top panel) or CEP-HSAfollowed by treatment with 10 μg C1Q (right panel) for a further 2 hrs.Oligomerisation of ASC-YFP was observed by speck formation, originalmagnification ×60. (d) IL-1β (left panel) and TNF-α (right panel)production in WT and Nlrp3^(-/-) BMDCs primed with LPS (3 h) andactivated with C1Q (16 h) at increasing doses (2.5, 5 and 10 μg C1Q).(e) IL-1β, IL-18 and IL-6 production in human PBMC primed with 100 ngml^(-/-) LPS overnight and activated with 5 μg ml C1Q for 6 h, with theaddition, 1 h before C1Q treatment of increasing doses (10 fold) of ZVAD(caspase-1 inhibitor). All ELISA data are representative of a minimum of3 separate experiments carried out in triplicate.

FIG. 4: Cleaved caspase-1 p10 co-localizes with activated macrophages inCEP-MSA immunized mice: (ac) Immunostaining of retinal cryosections ofCEP-MSA immunized mice showing localisation of F4/80 positivemacrophages (a) to regions of the choroid (b) extending from the choroidtowards Bruch's membrane and (c) present above the RPE in the outersegments (OS) and outer nuclear layer (ONL) of the retina. (a,b) Toppanel (c) left-hand panel, differential interference contrast (DIC)image, (a,b) bottom panel (c) right-hand panel, fluorescent image(F4/80-red, DAPI-blue). (d,e) Co-labelling of retinal cryosections ofCEP-MSA immunized mice with caspase-1 p10 (red) and F4/80 (green)showing co-localisation in (d) a macrophage present within andtranscending the choroid/Bruch's membrane and (e) a macrophageprotrusion in the OS of the retina. (f) Co-labelling of retinalcryosections of CEP-MSA immunized mice show co-localisation of NLRP3(red) and F4/80 (green). (g) High magnification of NLRP3 and F4/80staining.

FIG. 5: NLRP3 is protective against laser-induced CNV lesion formationin an IL-1β independent manner: (a) Laser induced CNV in WT (top leftpanel), Nlrp3^(-/-) (top middle panel) and IL1r1^(-/-) (top right panel)mice showing CNV development 6 days post laser burn. 3-D re-constructedimages of confocal Z-stacks from WT (bottom left panel), Nlrp3^(-/-)(bottom middle panel) and Il1r1^(-/-) (bottom right panel). CNV volumerendering (Bar chart). (b) Electroretinographic (ERG) analysis of rodand cone function of Nlrp3^(-/-) and IL1r1^(-/-) mice. (c)Immunostaining showing localization of activated macrophages(F4/80-green) to the site of laser induced injury in Nlrp3^(-/-) mice.(d) Immunostaining of WT (left hand panel) or Nlrp3^(-/-) (right handpanel) retinal cryosections 3 hours post injury, for cleaved caspase-1(red).

FIG. 6: NLRP3 confers its protection against CNV lesion formationthrough its role in IL-18 production, which in turn regulates VEGFlevels: (a) Electroretinographic (ERG) analysis of rod and cone functionof Il18^(-/-) mice. (b) Laser induced CNV in IL18^(-/-) mice showing CNVdevelopment 6 days post laser burn (left hand panel). 3-D re-constructedimages of confocal Z-stacks (right hand panel). CNV volume rendering(Bar chart). (c) CNV volumes were significantly increased compared to WTmice (FIG. 5) (*P=0.0292). The production of VEGF was assayed by ELISAin (d) ARPE-19 cells and (e) Mouse brain microvascular endothelial cells(B.end3) treated with increasing doses of IL-18 for 24 hrs or leftuntreated. ELISA data are representative of a minimum of 3 separateexperiments carried out in triplicate.

FIG. 7: (a) Western blot of NLRP3 expression in ARPE-19 cells (leftside) and THP1 cells (right side), equal amounts of protein were loadedas determined by BCA assay. (b) ARPE-19 cells were primed with variousTLR ligands; 100 ng/ml LPS, or 2 μg/ml Pam3Cys, or 25 μg/ml Poly(I:C),or 1 μg/ml R848, or 5 μg/ml CpG-ODN and either left un-treated oractivated with ATP for a further hour. IL-1β production was thenmeasured.

FIG. 8: a) RPE soup was observed under a light microscope to containretinal/RPE material produced following isolation of drusen. b) Thismaterial (100 ng/ml, 250 ng/ml and 500 ng/ml) was added to LPS primedPBMCs but caused no increase in IL-1(3 levels, or c) IL-18 levels. d)IL-6 expression was un-changed with increasing doses of RPE soup. e) RPEsoup elicited no change in IL-1β levels in WT or Nlrp3^(-/-) mouseBMDCs. f) There were no differential changes in IL-6 expression betweenthe experimental groups.

FIG. 9: Densitometric analysis of Caspase-1 P10 Western blot followingtreatment of THP1 cells with drusen.

FIG. 10: a) IL-1β levels were significantly increased in LPS primed WTand Tlr2^(-/-) BMDMs activated by ATP b) IL-6 levels were notsignificantly different.

FIG. 11: Densitometric analysis of Caspase-1 P10 Western blot followingtreatment of THP1 cells with increasing doses of C1Q.

FIG. 12: Zeta potential measurement of C1Q in solution shows a zetaaverage of 11.8 mV.

FIG. 13: IL-1β, IL-18 and IL-6 production in human PBMC primed with 100ng/ml LPS overnight and activated with 5 μg/ml C1Q for 6 h, with theaddition, 1 h before C1Q treatment of increasing doses (10 fold) of (a)DPI (ROS inhibitor), (b) Bafilomycin (inhibits lysosomal acidification),(c) CA-074 Me (cathepsin B inhibitor).

FIG. 14: CD68 staining (red) in a CEP-MSA immunized mouse retina, showedpositive cells in the sclera and outer segments (OS) of the retina.

FIG. 15: CEP-MSA immunized mouse retinal cryosections were stained forNLRP3 and positive immunoreactivity was observed in the (a) outersegments of the retina, (b) retinal pigment epithelium (RPE), (c) cellswithin the sclera and (d) cells within the choroid.

FIG. 16: F4/80 (left panel, red) and caspase-1 p10 (middle panel, green)co-localized to the site of laser induced CNV in WT mice (right panel)

FIG. 17: IL-18 was observed in at the site of laser induced injury in WTmice 24 h post injury (left panel-red staining). This staining was notevident at the site of injury in Nlrp3^(-/-) mice (right panel-redstaining).

FIG. 18 (a) Neutralizing IL-18 (1 μg) antibody injected post laserinduced CNV significantly increased CNV size in WT mice as measured byepifluorescent microscopy, (b) Confocal, Z-stack 3-D rendered image ofCNV. (c) Significantly increased CNV's were observed in WT mice injectedwith 1 ug IL-18 neutralizing antibody post laser injury compared to shaminjected mice (*P=0.0368).

FIG. 19 Flow cytometry analysis of (a) BMDC's and (b) BMDM's stained forCD11c and CD11b.

FIG. 20 (a) The RPE lies adjacent to the outer segments of thephotoreceptors. (b) A targeted thermal disruption of theretina/RPE/Bruch's membrane/choroid complex with a 532 nm laser causinga 50 μm diameter injury, Griffonia simplicifolia isolectin-Alexa-568(red) and Phalloidin-Alexa-488 (Green).

EXAMPLES Materials & Methods Drusen Isolation

Drusen and minor amounts of Bruch's membrane were isolated as previouslydescribed (8) from six AMD donor eyes (88M, 91 F, 97M, 85F, 85M and 80M)for use in these experiments.

CEP-Albumin Production

Human Serum Albumin (Sigma Aldrich, USA) was adducted with CEP aspreviously described (54).

ELISA Analysis

ELISA's were used to quantify cytokines in supernatants from the variousexperimental groups used throughout this study. IL-1β (RnD Systems),IL-18 (MBL International), IL-6 (RnD Systems), TNF-α (RnD Systems) andVEGF (RnD Systems) were analyzed throughout. All ELISA's were conducteda minimum of 3 times in triplicate. Inhibitors used during this studywere added at the following highest concentrations 1 h prior toinflammasome activation: 1 μg/ml of caspase-1 inhibitor VI (Calbiochem),5 μM cytochalasin D (Sigma Aldrich, Ireland), 10 μM CA-074 Me (cathepsinB inhibitor) (Sigma Aldrich, Ireland), 10 μM DPI (Sigma Aldrich,Ireland).

Western Blot Analysis

Generally, antibodies specific for caspase-1 (Santa-Cruz Biotech),beta-actin (Abcam), NLRP3 (Sigma Aldrich, Ireland), TLR-4 (Santa-CruzBiotech) were incubated on membranes overnight at 4° C. Membranes werewashed with TBS, and incubated with a secondary antibody against rabbit(IgG) with Horse-Radish-Peroxidase (HRP) conjugates (1:2500)(Sigma-Aldrich, Ireland), or mouse (IgG) (1:1000), (Sigma-Aldrich,Ireland), for 3 hours at room temperature. Immune complexes weredetected using enhanced chemiluminescence (ECL). All Western blots wererepeated a minimum of 3 times.

Cell Culture

ARPE-19 cell line (ATCC CRL 2302) were obtained from LGC promochem, THP1cells and primary isolated human peripheral blood mononuclear monocytes(PBMCs) were used for in vitro inflammasome activation assays. Cellswere cultured at 37° C., 5% CO₂, 95% air in a 1:1 mixture of Dulbecco'smodified Eagle's medium (DMEM) and Ham's F12 medium with 1.2 g/L sodiumbicarbonate, 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate(Sigma Aldrich) with 10% foetal calf serum (FCS). BMDCs and BMDMs werealso isolated from WT, NLRP3-/-, TLR-2-/-, C3H/HeN and C3H/HeJ mice on acongenic C57/B16 background. BMDCs and BMDMs were stained withanti-CD11c-APC and anti-CD11b-PeCy7. Cells were gated on live singlecells and expression of CD11c and CD11b was assesed by flow cytometry(FIG. 19). Mouse bEnd.3 microvascular endothelial cells were grown onfibronectin (Sigma Aldrich Ireland) coated tissue culture flasks in DMEMcontaining Glutamax and 10% FCS.

ASC Speck Formation Analysis.

Immortalized BMDMs (Gift from Dr Eicke Latz, University of Bonn)expressing yellow fluorescent (YFP) proteinlabelled ASC were primed withLPS, HSA or CEP-HSA, then activated with drusen or C1Q for either 3 or 6hrs respectively. Live cell imaging of speck formation was undertakenusing a temperature and CO₂ regulated confocal laser scanning microscopy(Olympus FluoView TM FV1000).

CEP-MSA Immunization

We used standard mouse immunization protocols (55). We anesthetized micewith ketamine-xylazine in PBS (80-90 mg/kg ketamine, 2-10 mg/mlxylazine). We used 200 μg of CEP-MSA in CFA or IFA (Difco Labs) forinitial and all booster doses as described previously (18).

Murine Models of Choroidal Neovascularisation (CNV)

All animal experiments conducted during the course of this work adheredto the Association for Research in Vision and Ophthalmology (ARVO)standards and all relevant national and institutional approvals wereobtained prior to commencement of the work. CNV, in which the vascularbed proliferates into the retina, mimicking neovascular AMD, was inducedin mice using a green 532 nm

Index Iris laser (532 nm, 140 mW, 100 mSec, 50 μm spot size, 3 spots pereye) incorporating a microscopic delivery system as described previously(21). This technique was used to induce CNV in Nlrp3-/-, IL1r1-/-,IL-18-/- and WT mice, and in each experimental assay animals were gendermatched. In tandem, we also directly injected, intra-vitreally postlaser burn, neutralizing antibodies directed against IL18 (Abcam). Micewere sacrificed 6 days post experiment and the neural retina wasremoved. Eye-cups were then incubated with aGriffonia-simplicifolia-isolectin-Aleax-568 molecule (Molecular Probes)(1:300) overnight at 4° C. and CNV's assessed by confocal microscopy(FIG. 20 a,b).

Indirect Immunostaining of Retinal Flatmounts and Retinal Cryosections

Indirect immunostaining was used to analyse activated macrophages andcleaved caspase-1, present in the neural retina in the animal models ofAMD. Antibodies against F4/80, CD68 (Abcam) for activated macrophages,and caspase-1 (P10) (Santa Cruz Biotech), NLRP3 (Santa Cruz Biotech andAbcam) and IL18 (Abcam) were used in conjunction with confocal laserscanning microscopy (Olympus FluoView TM FV1000).

Statistical Analyses

Statistical analysis was performed using Student's T-test, withsignificance represented by a P value of ≦0..05 when 2 individualexperimental groups were being analysed. For multiple comparisons, aswas the case in the ELISA analyses, ANOVA was used with a Tukey-Kramerpost-test and significance represented by a P value of ≦0.05.

Results

The RPE is a monolayer of cuboidal cells located between the outerretina and choroid. This melanized neuroepithelium has numerousfunctions including a) the adsorption of scattered and reflected light,b) the formation of the outer blood-retinal barrier (oBRB) and c) theremoval by phagocytosis of the effete tips of the photoreceptor outersegments (22). Proteomic and immunohistochemical analysis of drusen haveidentified virtually every protein involved in the complement cascade,proteins found in amyloid deposits as well as a number of crystallins,proteins synthesized in response to stress (23, 24). Considering therecent discovery that host-derived particulate matter such ascholesterol crystals and amyloid deposits (25, 26) can activate theNLRP3 inflammasome, we were interested to determine whether drusen couldalso initiate the activation of the inflammasome.

Drusen Activates the NLRP3 Inflammasome

Fundus photography of an unaffected eye compared to those of individualswith either dry or wet AMD (FIG. 1 a). Punctate light deposits in thefundus images represent drusen accumulation in both dry and wet AMD,with sub-retinal CNV apparent in the wet AMD photograph. Isolated drusenwas sonicated in order to dissociate the sample into small particulatematter (FIG. 1 b). SDS-PAGE analysis of the drusen sample showed acohort of high molecular weight proteins greater than 60 kDa (FIG. 1 c).The inflammasome is a multimeric protein complex. Caspase-1 is thecysteine protease activated in the inflammasome complex to cleavepro-IL-1β and pro-IL-18 into their mature forms. Activation of caspase-1requires the protein ASC which forms oligomers creating a platform forthe multimeric complex. Normally ASC is evenly distributed throughoutthe cell, but once activated ASC aggregates to a single point, known asa “speck”. BMDMs that stably express a yellow fluorescent proteinlabelled-ASC (YFP-ASC) were primed with LPS and treated with drusen ortransfected with Poly(dAdT) (positive control). ASC-YFP is difficult todiscern in macrophages treated with LPS alone (FIG. 1 d, left panels),however, in LPS primed macrophages activated with drusen, the formationof intense single fluorescent specks are clearly evident, indicative ofASC oligomerisation.

It is thought that the inflammatory response associated with AMD hasboth a local and systemic component. We initially tested the ARPE-19cell line for the presence of NLRP3 and for their ability to produceIL-1β in response to a range of TLR ligands and activation with ATP. Wefound that while ARPE-19 cells express NLRP3 the levels of IL-1β were atthe lower limit of assay sensitivity (FIG. 7). Peripheral myeloid cellsare the primary source of IL-1β and IL-18, with their ability to accessthe retina in AMD, we hypothesised that these cells would be key cellsof interest in our system. Human peripheral blood mononuclear cells(PBMCs) produced IL-1β and IL-18 in response to activation with druseneven at very low concentrations (FIG. 1 e,f). We used RPE material thatwas produced during the dissection of drusen from AMD eyes as a controlfor these experiments (FIG. 8). Immunoblot analysis of caspase-1expression in THP-1 cell lysates post-treatment with drusen confirmedincreased levels of cleaved caspase-1 p10 (FIG. 1 g and FIG. 9).Together, these results demonstrate that drusen from AMD donor eyes canactivate caspase-1 and the ASC inflammasome complex, which in turnresults in IL-1β and IL-18 production in PBMCs.

We reasoned that NLRP3 was the likely sensor for drusen-inducedinflammasome activation as it is required for inflammasome activation byparticulate matter. We isolated bone marrow from both wild type (WT) andNLRP3-deficient (Nlrp3^(-/-)) mice and cultured bone marrow derivedmacrophages and dendritic cells (BMDMs and BMDCs). Both WT BMDMs andBMDCs produced significant levels of IL-1β in response to drusen,conversely Nlrp3^(-/-) BMDMs and BMDCs (FIG. 1 h,i, left panels) wereunable to promote the production of mature IL-1β in response to drusen.Levels of IL-6 and TNFα were unaltered by the presence of drusen,indicative of a specific effect on IL-1β production (FIG. 1 h,i rightpanels). These results demonstrate that AMD drusen are capable ofactivating the NLRP3 inflammasome.

CEP-Adducted Human Serum Albumin Primes the NLRP3 Inflammasome

Up to 65% of the proteins that have been identified in drusen were foundin drusen isolated from both AMD and normal donors. However, oxidativeprotein modifications have also been observed in drusen, includingcarboxyethyl pyrrole protein adducts. Cumulative oxidative damagecontributes to aging and has long been suspected of contributing to thepathogenesis of AMD (27, 28, 29). Carboxyethyl pyrrole (CEP) adducts areuniquely generated from the oxidation of docosahexaenoate(DHA)-containing lipids and are significantly more abundant on drusenand serum of AMD subjects (19). Recently, carboxyalkylpyrroles, amongthem CEP, have been shown to be recognized by Toll-like receptor 2(TLR2) on endothelial cells (30). Given that TLR2 activation would primecells to induce pro-IL-1β, pro-IL-18 and NLRP3 we hypothesised that CEPadducted proteins in drusen and on Bruch's membrane could present anovel priming agent.

To test this we primed PBMCs with increasing concentrations ofCEP-adducted HSA or HSA alone and activated the cells with ATP. IL-1βlevels increased with increased concentrations of CEP-HSA, but nochanges were observed in cells primed with HSA alone (FIG. 1 a). WTBMDMs primed with CEP-HSA and activated with ATP also produced IL-1β, aneffect not observed in Nlrp3^(-/-) mice (FIG. 2 b). In order toascertain whether CEP-HSA was priming the cells through TLR2 activation,we primed WT and TLR2^(-/-) BMDMs with HSA or CEP-HSA and activated withATP. ATP activation induced IL-1β increases in WT but not TLR2^(-/-)BMDMs primed with CEP-HSA. Furthermore, no IL-1β induction was observedin BMDMs primed with HSA prior to activation, again confirming that itis the CEP modification that infers the ability to activate TLR2 (FIG. 2c). IL-6 levels are equivalent between CEP-HSA treated WT cells,confirming the specificity of the response for IL-1β (FIG. 2 d). IL-1βlevels were measured in LPS primed WT and TLR2^(-/-) BMDMs activated byATP to ensure TLR2 BMDMs were responding optimally (FIG. 10). To ensureour CEP-HSA was not LPS contaminated we isolated BMDMs from C3H/HeN andC3H/HeJ mice. C3H/HeJ mice carry a mutation in their Tlr4 gene whichrenders them un-responsive to LPS (31). C3H/HeJ BMDMs produced IL-1β inresponse to ATP when primed with CEP-HSA but not LPS (FIG. 2 e),indicating that our CEP adduct is LPS-free and primes the inflammasomethrough TLR2 ligation. TNF-α was detected in LPS primed WT C3H/HeN BMDMsbut not CEP primed cells (FIG. 2 f). We further examined the ability ofCEP to prime the NLRP3 inflammasome by measuring ASC-YFP speck formationin CEP-treated BMDMs. Focused ASC-YFP specks were observed in BMDMsprimed with CEP-HSA and activated with drusen (FIG. 2 g, top panel).Drusen alone appeared to be able to cause the oligomerisation of ASC(FIG. 2 g, bottom panel), implying that alone, drusen could initiate theformation of the multi-protein platform for inflammasome activation.However, we were unable to consistently detect IL-1β increases whenPBMCs or BMDM/BMDCs were treated with drusen alone and assayed by ELISA.

Drusen Component Complement Factor C1Q, Activates the Inflammasome

Although drusen can distort and eventually damage the retina as in GA(29), not all people presenting with drusen develop vision loss,therefore it is conceivable that in addition to the particulate natureof drusen causing mechanical insult to the RPE, some component(s) ofdrusen may be involved in the activation of the inflammasome in a morespecific manner. We elected to study C1Q, the primary initiatingcomponent of the classical complement pathway, which has been identifiedin drusen (32). Since C1Q is an effector of the innate immune systemwith the potential to be extremely damaging to host tissue, its presencein drusen is indicative of an earlier or ongoing inflammatory insult. Wedirectly evaluated the ability of C1Q to activate the NLRP3inflammasome. Addition of C1Q alone to BMDMs did not cause theproduction of IL-1β, however cells that were primed with CEP-HSA beforethe addition of C1Q produced significant levels of IL-1β (FIG. 3 a, leftpanel). Secretion of the pro-inflammatory cytokine TNFα remainedunchanged upon addition of C1Q) to CEP-HSA primed BMDMs (FIG. 3 a, rightpanel), indicating that C1Q is specifically activating the inflammasomeand is not involved in the up-regulation of pro-inflammatory cytokinesin general. We observed cleaved caspase-1 p10 in THP1 human monocyticcells activated with C1Q (FIG. 3 b, FIG. 11) and further establishedthat C1Q could cause ASC oligomerisation as YFP-ASC specks can be seenin concentrated focal points within the cells activated with C1Q afterpriming with either LPS (FIG. 3 c, top right-panel) or with CEP (FIG. 3c, bottom right-panel).

WT BMDCs treated with C1Q did produce a significant level of IL-1β,however Nlrp3^(-/-) BMDMs failed to produce IL-1β in response to C1Qactivation (FIG. 3 d, left), levels of TNFα remained unchanged (FIG. 3d, right). To confirm the role of caspase-1, we added a caspase-1inhibitor, ZVAD, to human PBMC before C1Q activation. Caspase-1inhibition decreased both IL-1β and IL-18 production in a dose dependentmanner (FIG. 3 e). Together these results show that C1Q can act as adanger signal sensed by the NLRP3 inflammasome. All C1Q isolated fromhuman blood and C1Q found in drusen has a propensity to aggregate and wehave shown this following zeta-potential analysis of a solution of C1Q,we believe this is a key factor in how C1Q can activate the NLRP3inflammasome (FIG. 12)

C1Q Inflammasome Activation Involves the Phagolysosome

Deposits of C1Q along with other complement factors have been shown tobe associated with, or components of, amyloid structures (33, 34). It istherefore likely that C1Q as a component of drusen would result in itsaggregation and assist macrophages as they attempt to phagocytose theseparticulate deposits. The mechanisms leading to NLRP3 inflammasomeactivation are still a matter of debate and may depend on the stimulus.One mechanism involves the phagocytosis of particulate structuresleading to lysosomal rupture and release of lysosomal contents (35).Another proposed mechanism involves the production of reactive oxygenspecies (ROS) which lead to the activation of the NLRP3 inflammasome viaROS-sensitive TXNIP protein (36). To determine if C1Q induction of ROS(37,38) was responsible for inflammasome activation we treated PBMC withthe NADPH oxidase inhibitor DPI prior to C1Q activation. Inhibition ofROS by DPI had no effect on C1Q induced IL-1β release (FIG. 13 a). Thealternative mechanism proposed is that lysosomal instability leads tothe leakage of the lysosomal-exopeptidase, cathepsin B, into the cytosolwhich is sensed by the components of the inflammasome leading to itsassembly (35). To determine the role of the phagolysosome in theactivation of the inflammasome by C1Q we used bafilomycin A, aninhibitor that blocks the vacuolar H⁺ ATPase system necessary forlysosomal acidification and the cathepsin B inhibitor CA-074 Me.Inhibition of either vacuolar H⁺ ATPase or cathepsin B restricted C1Qactivated production of IL-1β and IL-18 with no effect on IL-6production (FIG. 13 b, c). This directly implies that C1Q alters thephagolysosomal process to trigger NLRP3 activation.

NLRP3 Inflammasome is Active in CEP-MSA Immunized Mice

We sought to determine whether the inflammasome was involved in thepathology of a well characterised model of dry AMD, the CEP-MSAimmunised mouse model. This animal develops AMD-like lesions in itsretina and RPE following immunization with CEP-MSA. We analysed retinalsections of CEP-MSA immunized mice, for the presence of activatedmacrophages (F4/80 and CD68 staining), caspase-1 p10 and NLRP3.Activated macrophages were observed to be present within the choroid andBruch's membrane (FIG. 4 a,b, FIG. 14), We also observed infiltratingmacrophages above the RPE in the outer segments of the retina (FIG. 4c). Staining of these sections showed co-localisation of F4/80 withcleaved caspase-1 p10 (FIG. 4 d,e) and NLRP3 (FIG. 4 f,g, FIG. 15).

NLRP3 Protects Against Exacerbated Laser-Induced CNV Development

A much used model for wet (exudative) AMD is laser induced CNV, which isalso an ideal model for sterile inflammation (39), likely due to theinduction of a necrotic microenvironment within the tissue. Necroticcells are known to trigger a sterile inflammatory response through theNLRP3 inflammasome (17). We hypothesised that the NLRP3 inflammasome mayplay a key role in CNV development in response to localised tissueinjury. In order to test our hypothesis we administered focal laserburns to the retinas of WT, Nlrp3^(-/-) and Il1r1^(-/-) mice andassessed CNV volumes. Surprisingly we found significantly more CNVdevelopment and sub-retinal haemorrhaging in Nlrp3^(-/-) mice whencompared with WT and IL1r1^(-/-) mice (FIG. 5 a). 3D Z-stack confocalvolume rendering of CNVs confirmed a significant increase in CNV volumein Nlrp3^(-/-) mice 6 days post injury (FIG. 5 d, histogram).Electroretinographic (ERG) analysis confirmed both knockout mice havefunctional rod and cone responses pre-injury (FIG. 5 b). We observedactivated macrophage infiltration (positive F4/80 immunoreactivity) atthe lesion site in Nlrp3^(-/-) mice (FIG. 5 c), however, cleavedcaspase-1 and IL-18 were only evident at the injury site of WT mice andwere notably absent in Nlrp3^(-/-) mice (FIG. 5 d, FIG. 16, 17). Thesefindings describe a role for the NLRP3 inflammasome in the sterileinflammatory response observed in this animal model of CNV and pointtowards IL-18 as a regulator of CNV development.

NLRP3 Confers Protection Against CNV Lesion Formation Through IL-18

In order to confirm a role for IL-18 in NLRP3-mediated protectionagainst exacerbated CNV development, we administered laser induced CNVsin IL18^(-/-) mice. These mice were observed to have normal retinalfunction (FIG. 6 a) as assessed by ERG analysis. Laser induceddisruption of Bruch's membrane and CNV volume quantification inIL18^(-/-) mice 6 days post injury showed markedly increased lesions(FIG. 6 b) compared to WT CNVs (FIG. 6 c). Intravitreally injected IL-18neutralising antibodies subsequent to laser induced CNV also resulted insignificantly increased CNV development (FIG. 18).

We reasoned that IL-18 might confer its protection via the regulation ofVEGF synthesis. To test this hypothesis we treated ARPE19 cells and amouse brain microvascular endothelial cell line (bEnd.3) withrecombinant IL-18 and subsequently analysed VEGF levels in the growthmedium. IL-18 significantly decreased levels of VEGF secreted by bothARPE-19 cells and bEnd.3 cells (FIG. 6 d,e). These findings directlyimplicate a role for IL-18 in the regulation of VEGF expression andlikely explain the exacerbated CNVs in Nlrp3^(-/-) and IL18^(-/-) mice.

Conclusion

Our studies have shown that drusen isolated from AMD donor eyes canactivate the NLRP3 inflammasome. Furthermore, we show thatcarboxyethyl-pyrrole (CEP), an oxidative stress related proteinmodification commonly found decorating drusen proteins, can prime theinflammasome. In tandem, we show that the complement component C1Q canactivate the NLRP3 inflammasome in a caspase-1 and phagolysosomedependent manner. We observed activated caspase-1 and NLRP3 inmacrophages surrounding the drusen-like lesions associated with CEP-MSAimmunised mice, an accepted model of dry AMD. We also found that acommonly used animal model of wet AMD, is dependent on NLRP3 activation,but unexpectedly in the absence of NLRP3, CNV development wasexacerbated. We implicate IL-18 as a key regulator of pathologicalneovascularisation and suggest a protective role for the NLRP3inflammasome in the development of AMD.

Our observations have major implications in regard to prevention of AMD.Current antibody-based therapies target advanced forms of AMD byinhibiting the bioactivity of VEGF. However, direct and regularintraocular injection of these monoclonal antibodies (Lucentis® andAvastin®) carry the risk of retinal detachment, haemorrhage andinfection.

We have shown that drusen isolated from AMD donor eyes can activate theNLRP3 inflammasome. AMD drusen is composed of a collection of proteindeposits, many of which are adducted to CEP. Due to its particulatenature, it is possible that drusen from normal donor eyes may alsoinduce inflammasome activation, however its levels in the retina bydefinition, are lower than AMD drusen and the biochemical compositionsare different. These differences are likely important for theprogression of AMD. A comparison of control and AMD drusen in relationto inflammasome activation has, however, yet to be fully elucidated.

We have demonstrated that CEP-HSA can prime the inflammasome throughTLR2 activation providing us with a naturally occurring priming agentthat accumulates at focal points at high levels within the AMD eye. Inthe case of NLRP3, the danger signal is usually particulate andextra-cellular in nature. C1Q, a component of drusen, has been shown toaggregate in an amyloid-like fashion. We show that C1Q, isolated fromhuman blood, activates the NLRP3 inflammasome in a manner dependent onlysosomal acidification and cathepsin B.

The sterile inflammatory response that occurs in AMD is likely a resultof the focal necrosis that occurs in the RPE cells sub-adjacent toexcessive drusen accumulation. Drusen accumulation in Bruch's membraneis a hallmark feature and diagnostic indicator of early AMD developmentand is thought to be central to the pathology of the disease. While wehave observed inflammasome activation in macrophages associated withAMD-like lesions in CEP-MSA immunised mice, our observations for thefirst time directly indicate a protective role for inflammatoryprocesses in the progression to CNV, the exudative form of AMD, anddirectly oppose current dogma directed at suppression of inflammatoryprocesses in disease prevention. Indeed it is now accepted that somelevel of inflammation, “para-inflammation”, may be beneficial to thehost. From a clinical perspective, while inflammatory processes havelong been associated with AMD pathology and disease development, wesuggest that global inhibition of inflammation in the retina in the caseof wet AMD would not be a sound therapy. Lending strength to ourobservations, the results of recent clinical trials of Infliximab(Remicade®) in individuals with wet AMD showed that in more than 50% ofthese subjects, symptoms were greatly exacerbated.

The NLRP3 inflammasome has also recently been shown to conferprotection, through IL-18 production, against experimental colitis andcolorectal cancer in mice.

Previous studies indicate that IL-18 plays an important role in retinalvascular development. Il-18^(-/-) mice showed angiectasis and vascularleakage, VEGF and bFGF levels were also up-regulated in the Il-18^(-/-)mouse retinas. Anti-angiogenic roles for IL-18 have also been observedin post-ischemic injury and in the inhibition of tumour angiogenesis.

Activation of the NLRP3 inflammasome by drusen suggest that a balancemay exist, whereby a certain focal level of drusen is tolerated due toits ability to induce IL-18 which in turn may act as an anti-angiogeniceffector, maintaining choroidal homeostasis in an inflammatorymicro-environment. It is likely that once a critical level of drusenaccumulates, its protective role is negated by excessive damage to thesurrounding tissues. Importantly, we have demonstrated thatdrusen-inducible inflammatory mediators are protective against CNVdevelopment and that it is the resultant NLRP3 mediated elevation ofIL-18 that prevents the downstream production of VEGF. Moreover, IL-18has been shown not to play a role in the development of experimentaluveitis, a more conventional model of inflammation, a finding which hasdirect implications for future forms of therapy deriving from ourfindings. Overall, our observations directly implicate NLRP3 as aprotective agent against the major disease pathology of AMD and suggestthat strategies aimed at producing or delivering IL-18 to the eye, mayprove beneficial in preventing the progression of CNV in the context ofwet AMD.

Supplementary Methods Clinical Evaluation

AMD subjects and un-affected individuals were assessed by a clinicalophthalmologist following informed consent. Best-corrected distancevisual acuity was measured using a Snellen Chart. Near vision wasassessed using Standard Test Type. The anterior segment of the eye wasexamined by slit-lamp biomicroscopy. Intraocular pressure was measuredby Goldmann Tonometry. Detailed funduscopic examination and colourfundus photography were carried out following pupillary dilation usingTropicamide (1%). Dry AMD was diagnosed by the presence of visualdistortion due to AMD-associated macular changes (drusen,hyperpigmentation, hypopigmentation of the RPE or geographic atrophy).Wet AMD was diagnosed by clinical examination supplemented byfluorescein angiographic photography to illustrate CNV.

ERG Analysis of Mice

Mice were dark-adapted overnight and prepared for electroretinographyunder dim red light. Pupillary dilation was carried out by instillationof 1% cyclopentalate and 2.5% phenylephrine. Animals were anesthetizedby intraperitoneal (IR) injection of ketamine (2.08 mg per 15 g bodyweight) and xylazine (0.21 mg per 15 g body weight). Standardisedflashes of light were presented to the mouse in a Ganzfeld bowl toensure uniform retinal illumination. The ERG responses were recordedsimultaneously from both eyes by means of gold wire electrodes (RolandConsulting Gmbh) using Vidisic (Dr Mann Pharma, Germany) as a conductingagent and to maintain corneal hydration. Reference and ground electrodeswere positioned subcutaneously, approximately 1 mm from the temporalcanthus and anterior to the tail respectively. Body temperature wasmaintained at 37° C. using a heating device controlled by a rectaltemperature probe. Responses were analysed using a RetiScan RetiPortelectrophysiology unit (Roland Consulting Gmbh). The protocol was basedon that approved by the International Clinical Standards Committee forhuman electroretinography. Cone-isolated responses were recorded using awhite flash of intensity 3 candelas/m⁻²/s presented against arod-suppressing background light of 30 candelas/m⁻² to which thepreviously dark adapted animal has been exposed for 10 minutes prior tostimulation. The responses to 48 individual flashes, presented at afrequency of 0.5 Hz, were computer averaged. Following the standardconvention, a-waves were measured from the baseline to a-wave trough andb-waves from the a-wave trough to the b-wave peak.

The invention is not limited to the embodiment(s) described herein butcan be amended or modified without departing from the scope of thepresent invention.

The invention will now be described by the following first set ofembodiments.

1. Inflammatory mediators that are protective against degenerativeretinal conditions

2. Inflammatory mediators that are components of the NLRP3-inflammasome

3. NLRP3 Inflammasome components that is pro-inflammatory Interleukin-18(IL-18)

4. Inflammasome components according to any of embodiments 2 to 3 foruse in the treatment of degenerative retinal conditions involving thedrusen and anaphylatoxin-induced choroidal-neovascularisation (CNV)

5. Inflammasome components according to embodiment 4 wherein thedegenerative retinal condition is age-related macular degeneration(AMD), including wet and dry AMD.

6. NLRP3-Inflammasome according to any of the preceding embodimentswherein the activity of IL-18 is maintained and/or stimulated

7. Use NLRP3 Inflammasome induced IL-18, variants or a part thereof inthe manufacture of a medicament for the treatment of degenerativeretinal conditions involving the drusen and anaphylatoxin-inducedchoroidal-neovascularisation (CNV) wherein the treatment involves thedelivery of NLRP3 Inflammsome activators, such as IL-18, to bone marrowor bone marrow derived cells or tissues and/or directly to ocular cellsor tissues.

8. Use according to embodiment 7 comprising viral-mediated delivery ofIL-18

9. A method for retardation of the progression of degenerativeretinopathies involving maintaining or stimulating the activity of IL-18

10. Use of NLRP3-Inflammasome induced mediators, preferablypro-inflammatory cytokine IL-18, as a biomarker to indicate the risk ofdeveloping diseases involving drusen and anaphylatoxin-inducedchoroidal-neovascularisation associated diseases, such wet or dry AMD.

11. A method for determining the risk of development or progression ofdiseases drusen and anaphylatoxin-induced choroidal-neovascularisationassociated diseases, such wet or dry AMD, in a subject, using-Inflammasome induced mediators, preferably pro-inflammatory cytokineIL-18, as a biomarker the method comprising;

-   -   obtaining the level of circulating IL-18 levels in the subject;    -   comparing the level of IL-18 levels to a reference, wherein the        subject's risk of development or progression of the disease        drusen and anaphylatoxin-induced choroidal-neovascularisation is        based upon the level of IL-18 levels in comparison to the        reference.

The invention will now be described by the following second set ofembodiments.

-   1. Inflammatory mediators, preferably components or substrates of    the NLRP3-inflammasome, for use in the treatment of degenerative    retinal conditions involving the drusen and anaphylatoxin-induced    choroidal-neovascularisation.-   2. Inflammatory mediators, components or substrates for use    according to embodiment 1 wherein the retinal condition is    age-related macular degeneration.-   3. Inflammatory mediators, components or substrates for use    according to embodiment 2 wherein the age-related macular    degeneration is wet age-related macular degeneration or dry    age-related macular degeneration.-   4. Inflammatory mediators, components or substrates for use    according to any of the preceding embodiments wherein the component    of the NLRP3-inflammasome is pro-Interleukin-18 (pro IL-18) or    Interleukin-18 (IL-18).-   5. Inflammatory mediators, components or substrates for use    according to any of the preceding embodiment s for use in a method    wherein pro-Interleukin-18 (pro-IL-18) or Interleukin-18 (IL-18) is    administered to a subject prior to the development of neo-vascular    disease or in early-stage neo-vascular disease.-   6. Inflammatory mediators, components or substrates for use    according to any of the preceding embodiment s in controlling,    maintaining or stimulating Interleukin-18 (IL-18) expression in a    subject at risk of developing age-related macular degeneration.-   7. Pro-Interleukin-18 (pro-Il-18) for use according to any of the    preceding embodiments for use in controlling    choroidal-neovascularisation (CNV) in a patient at risk of    developing wet age-related macular degeneration (AMD).-   8. Pro-Interleukin-18 (pro-Il-18) for use according to embodiment 7    wherein pro-Interleukin-18 (pro-IL-18) is delivered to the retina.-   9. Pro-Interleukin-18 (pro-Il-18) for use according to embodiment 8    wherein pro-Interleukin-18 (pro-IL-18) is delivered to the retina    systemically, by direct injection and/or by viral mediated delivery.-   10. Pro-Interleukin-18 (pro-Il-18) for use according to embodiment 8    or 9 wherein pro-Interleukin-18 (pro-IL-18) is delivered to the    retina by adeno-associated viral (AAV) mediated delivery.-   11. Pro-Interleukin-18 (pro-Il-18) for use according to embodiment    10 wherein an adeno-associated virus (AAV) expressing an inducible    pro-IL-18 is delivered to the retina.-   12. Pro-Interleukin-18 (pro-Il-18) for use according to any of the    preceding embodiments wherein the expression of caspase-1 in and    around retinal pigment epithelial (RPE) cells regulates the    processing of pro-IL-18 to IL-18 and the IL-18 has a protective    effect on choroidal neo-vascularisation (CNV) development.-   13. A recombinant viral gene delivery vector which directs the    expression of pro-IL-18 in a form suitable for the treatment of    ocular, preferably choroidal, neo-vascularisation.-   14. The recombinant viral gene delivery vector according to    embodiment 13 in a form for administration to the eye of a subject    at risk of developing AMD, preferably wet AMD.-   15. The recombinant viral gene delivery vector according to    embodiment 13 or 14 wherein the vector is in a form suitable for    delivery to the eye via sub-retinal or intra-vitreal injection.-   16. The recombinant viral gene delivery vector according to any of    embodiments 13 to 15 wherein the viral vector is an adeno-associated    virus (AAV), adenovirus or lentivirus gene delivery vector.-   17. The recombinant adeno-associated virus (AAV) gene delivery    vector according to embodiment 16 which directs the expression of    pro-IL-18 and is suitable for administration to the eye of a subject    at risk of developing AMD, preferably wet AMD.-   18. The recombinant AAV vector according to embodiment 17 wherein    the gene delivery vector is an AAV vector comprising nucleotides    encoding pro-Il-18 gene.-   19. The recombinant AAV vector according to embodiment 17 or 18    wherein the AAV is any one of AAV serotype 1 to 11, preferably    serotype 2, 8 or 9.-   20. The recombinant AAV vector according to any of embodiments 16 to    19 wherein the pro-IL-18 gene is flanked by AAV inverted terminal    repeats (ITRs).

21. A method of treating degenerative retinal conditions involving thedrusen and anaphylatoxin-induced choroidal-neovascularisation comprisingadministering inflammatory mediators, components or substrates of theNLRP3-inflammasome to a subject in need of treatment.

-   22. The method according to embodiment 21 for treating wet    age-related macular degeneration (AMD) comprising administering    pro-Interleukin-18 (pro-Il-18) to a subject at risk of developing    age-related macular degeneration.

1. A method of treating degenerative retinal conditions involving drusenand anaphylatoxin-induced choroidal-neovascularisation comprisingadministering inflammatory mediators, components, or substrates of theNLRP3-inflammasome to a subject in need thereof.
 2. The method accordingto claim 1 wherein the component of the NLRP3-inflammasome isInterleukin-18 (IL-18).
 3. The method according to claim 1 wherein theretinal condition is selected from the group consisting of age-relatedmacular degeneration, wet age-related macular degeneration, and dryage-related macular degeneration.
 4. The method according to claim 1wherein the component of the NLRP3-inflammasome is deliveredsystemically to said subject.
 5. The method according to claim 1 whereinthe component of the NLRP3 -inflammasome is delivered locally to saidsubject.
 6. (canceled)
 7. The method according to claim 1, wherein saidsubject is at risk of developing wet age-related macular degeneration(AMD), and wherein choroidal-neovascularisation (CNV) is controlled saidsubject.
 8. The method according to claim 1, wherein Interleukin-18(IL-18) is administered to said subject prior to the development in saidsubject of neo-vascular disease or early-stage neo-vascular disease. 9.The method according to claim 1, wherein said subject is at risk ofdeveloping wet age-related macular degeneration (AMD), furthercomprising controlling, maintaining or stimulating Interleukin-18(IL-18) expression in said subject.
 10. The method according to claim 1,wherein Interleukin-18 (IL-18) is delivered to at least one eye, retina,and/or choroid of said subject.
 11. The method according to claim 1,wherein Interleukin-18 (IL-18) is delivered to at least one retina ofsaid subject systemically, by injection and/or by viral mediateddelivery.
 12. The method according to claim 1, wherein Interleukin-18(IL-18) is delivered to the at least one retina of said subject byadeno-associated viral (AAV) mediated delivery.
 13. The method accordingto claim 1, wherein IL-18 has a protective effect on choroidalneo-vascularisation (CNV) development.
 14. A recombinant viral genedelivery vector which directs the expression of IL-18 in a form suitablefor the treatment of choroidal neo-vascularisation.
 15. The recombinantviral gene delivery vector according to claim 14 in a form foradministration to the eye of a subject at risk of developing age-relatedmacular degeneration, wet age-related macular degeneration and/or dryage-related macular degeneration.
 16. The recombinant viral genedelivery vector according to claim 14 wherein the vector is in a formsuitable for delivery to the eye by intraocular injection, sub-retinal,injection, intra-vitreal injection, retrobulbar injection,subconjunctival injection, and/or subtenon injection.
 17. Therecombinant viral gene delivery vector according to claim 14, whereinthe viral vector is an adeno-associated virus (AAV), adenovirus orlentivirus gene delivery vector.
 18. (canceled)
 19. (canceled) 20.(canceled)
 21. A method for treating a subject affected by or at risk ofdeveloping a disease involving drusen and anaphylatoxin-inducedchoroidal neo-vascularisation, the method comprising: a) obtaining thelevel of circulating IL-18 and/or IL-18 binding protein levels in saidsubject; b) comparing said IL-18 and/or IL-18 binding protein levels toa reference, wherein the subject's risk of development or progression ofthe disease drusen and anaphylatoxin-inducedchoroidal-neovascularisation is based upon the level of IL-18 levels incomparison to said reference; and c) administering inflammatorymediators, components, or substrates of the NLRP3-inflammasome to saidsubject based on said binding protein levels.
 22. The method accordingto claim 21 wherein the drusen and anaphylatoxin-induced choroidalneo-vascularisation is selected from wet or dry age-related maculardegeneration.
 23. The method according to claim 5 wherein local deliveryis selected from intraocular injection, sub-retinal injection,intra-vitreal injection, retrobulbar injection, subconjunctivalinjection and/or subtenon injection.
 24. The method according to claim1, wherein Interleukin-18 (IL-18) is delivered to at least one retina ofsaid subject by an adeno-associated virus (AAV) expressing an inducibleIL-18.